First strand cdna synthesis kit

Total mRNA from parental and CDV^R cells was isolated with the Quickprep mRNA purification kit (Amersham Biosciences) and converted to cDNA with the first-strand cDNA synthesis kit (GE Healthcare). The entire cDNA from each selected gene was amplified by PCR using specific primers. The PCR products were purified using PCR product purification kit (Roche) and directly sequenced using a cycle-sequencing kit (Dyenamic dye terminator kit; Amersham Biosciences), specific primers targeting both strands of the specific gene, and a capillary DNA sequencing system (MegaBACE 500; Amersham Biosciences). The data were assembled and compared to the DNA sequences obtained from reference sequences using Vector NTI software (Invitrogen). The primers used for the genotyping of UMP/CMPK1 and 2, and HPV oncogenes E6 and E7 are listed in Table S2.

For expression analyses, stigmas, corms, roots, leaves, stamens and tepals were dissected from ten plants and the same kind of tissues were pooled. Three independent pools of these tissues were used as three biological replicas. TRIzol reagent (Invitrogen) was utilized for RNA extraction. 1 µg of total RNA was used for first-strand cDNAs by reverse transcription (RT) using an oligo dT primer and a First-strand cDNA Synthesis Kit (GE Healthcare Life Sciences, Buckinghamshire, UK) according to the manufacturer’s instructions. The cDNAs were used as templates using gene-specific primers (Table S1). The cycling parameters of qPCR consisted of an initial denaturation at 94 °C for 5 min, 40 cycles at 94 °C for 20 s, 58 °C for 20 s, 72 °C for 20 s, and a final extension at 72 °C for 5 min. The assays were conducted in a StepOne™ Thermal Cycler (Applied Biosystems, Foster City, CA, USA) and analyzed using StepOne software v2.0 (Applied Biosystems, Foster City, CA, USA). DNA melt curves were created for each primer combination in order to confirm the presence of a single product. Gene expression was calculated using the 2^−ΔΔCT method. The expression data were normalized based on CsRP18S [8 (link),59 (link)].

NucleoZOL (Macherey‐Nagel) was used to perform total RNA extraction according to the manufacturer's recommendations. The First‐Strand cDNA Synthesis Kit (GE Healthcare, Chalfont St Giles, UK) was used to synthesize cDNA from 2 μg of total RNA, according to the manufacturer's instructions. The RT reaction mixture was diluted 1:4 and used as a cDNA template for qPCR. TaqMan quantitative PCR was carried out on a FX96 Thermocycler (Bio‐Rad). The PCR mixture contained TaqMan mix (Roche), 200 nM of primers (see Table S2 for their sequence), Universal Probe Library probe (100 μM, ThermoFisher Scientific) for the gene of interest (TaqMan Gene Expression Assays [Primers/probe], Life technologies), and 50 ng/μg of cDNA template. Reactions were performed in triplicate with the following program: 95°C 10 min, followed with 40 cycles of 95°C for 10 s, 59°C for 30 s. The relative amount of mRNA was calculated using the comparative Ct (ΔΔCT) method, following data normalization against GAPDH housekeeping gene.

Pellets from cells or zebrafish samples were collected and stored at −80°C until use. The samples were processed for RNA extraction by using an “RNeasy^® protect mini kit” (Qiagen, Hilden, Germany), as previously reported (Cecconi et al., 2019 (link)). The RNA samples were quantified using a Qubit™ RNA HS assay kit” (Invitrogen, Carlsbad, United States) and a Qubit 3 Fluorometer (Invitrogen by Thermo Fisher Scientific, REF Q3321). RNA (1 mg) was reverse-transcribed using the first-strand cDNA synthesis kit (GE Healthcare, Little Chalfont), as per the manufacturer’s instruction, and a SimpliAmp Thermal Cycler (Applied Biosystems by Thermo Fisher Scientific, REF A24812). RNA and cDNA samples were stored at −80°C until use.

Normal and tumoral human kidney tissues were provided by the biobank of the CHU of Lille. Phenol-chloroform total RNA extraction was performed with TriReagent (Sigma-Aldrich, Saint Louis, MO, USA). PhaseLockGel tubes (Eppendorf, Hamburg, Germany) were used for phase separation. cDNA was synthesized from 3 μg total RNA with the First-Strand cDNA Synthesis Kit (GE Healthcare, Chalfont St Giles, UK). The RT reaction mixture was diluted 1/60 and used as cDNA template for qPCR analysis. TaqMan quantitative PCR analysis was carried out on a LightCycler 2.0 System (Roche Applied Science). PCR mixtures contained LightCycler TaqMan mix, 200 nM primers, and 1.67 μl cDNA template in a 6.67-μl reaction volume. The PPIA or ACTB housekeeping gene, as indicated, was used in kidney samples for normalization of PLA2R1 mRNA expression in each sample type. Intron-spanning real-time PCR assays were designed with the ProbeFinder software (Roche Applied Science). All real-time PCR primers are listed in Supplementary Table 1.

cDNA synthesis was performed using First-Strand cDNA Synthesis Kit as per manufacturer’s instructions (GE Healthcare Bio-Science Inc. Baie d’Urfe, QC, Canada). Briefly, 20 μL of diluted RNA (concentrations ranged from 224 to 890 ng/μL) was heated at 65°C for 10 minutes in the GeneAMP polymerase chain reaction (PCR) System 2700 (Applied Biosystems, Foster, CA, USA). Once complete, 11 μL of the bulk first strand cDNA reaction mix, 1 μL of poly (dT) primer, and 1 μL of DTT solution was added. The reaction was incubated at 37°C for 60 minutes. The cDNA concentration was measured using a Gene Quant Pro RNA/DNA calculator (Biochrom Ltd, Cambridge, UK). All cDNA products were stored in −20°C for subsequent real time PCR use.